Ocorrência de seqüências relacionadas com a virulência e análise filogenética de estirpes comensais e patogênicas de Escherichia coli aviário (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.533540Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD50 of 4.0 × 105 CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD50 of 1.2 × 1012 CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes

A Fatal Bacteremia Caused by Hypermucousviscous KPC-2 Producing Extensively Drug-Resistant K64-ST11 Klebsiella pneumoniae in Brazil

We report a fatal bacteremia caused by Klebsiella pneumoniae in a 60–70-year-old patient from Brazil. The genomic analysis of three isolates (from blood culture, nasal and anal swabs) showed that the bacteremia was caused by a KPC-2 producing extensively drug-resistant K64-ST11 hypermucousviscous K. pneumoniae (hmKP) harboring several virulence and antimicrobial resistance genes. Although the isolates did not present virulence markers associated with hypervirulent K. pneumoniae (hvKP), they showed invasion and toxicity to epithelial Hep-2 cells; resistance to cell microbicidal mechanisms; and blood and human serum survival, evidencing their pathogenic potential. This study highlights the risk of infection caused by hmKp strains not characterized as hvKP as well as the clinical implications and difficulty of treatment, especially in elderly or immunocompromised patients

The potential of using E. coli as an indicator for the surveillance of antimicrobial resistance (AMR) in the environment

To understand the dynamics of antimicrobial resistance (AMR), in a One-Health perspective, surveillance play an important role. Monitoring systems already exist in the human health and livestock sectors, but there are no environmental monitoring programs. Therefore there is an urgent need to initiate environmental AMR monitoring programs nationally and globally, which will complement existing systems in different sectors. However, environmental programs should not only identify anthropogenic influences and levels of AMR, but they should also allow for identification of transmissions to and from human and animal populations. In the current review we therefore propose using antimicrobial resistant Escherichia coli as indicators for monitoring occurrence and levels of AMR in the environment, including wildlife.publishedVersio

Estudo comparativo dos fatores de virulencia de pseudomonas aeruginosa isoladas de fibrose cistica e outras infecções

Orientador: Domingos da Silva LeiteDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Fibrose Cística (FC) é uma doença genética, autossômica recessiva, caracterizada por anormalidade no transporte eletrolítico. Pseudomonas aeruginosa é um importante patógeno oportunista, freqüentemente encontrado em pacientes com FC. Nesses pacientes, a colonização inicial é feita com a bactéria na forma não mucóide que, posteriormente, converte-se à forma mucóide. O objetivo desse trabalho foi verificar comparativamente a produção de alguns fatores de virulência por P. aeruginosa em amostras mucóides e não mucóides isoladas de pacientes com FC e em amostras isoladas de pacientes não portadores de FC. O método para detectar hemolisina utilizou placas de ágar sangue contendo 5% de sangue de carneiro. A hidrólise da gelatina foi utilizada para detectar a produção de gelatinase e a produção de elastase foi observada em placas contendo 1 % de elastina. A presença de pili foi analisada pelo método de microhemaglutinação e a cápsula de alginato, pelo método de PCR para identificar os genes algD e algU. As linhagens não mucóides isoladas de pacientes com FC e aquelas isoladas de outros casos clínicos produziram as exoenzimas gelatinas e e elastase em maior freqüência que as linhagens mucóides. A produção de hemolisina e a presença dos genes algD e algU foi semelhante nos três grupos bacterianos. O gene algD foi encontrado em todas as linhagens estudadas e o ensaio de microhemaglutinação não foi satisfatório para detectar a presença de pili em P. aeruginosaAbstract: Cystic Fibrosis (CF) is a genetic disease, recessive autosomal, characterized by anomalies on eletrolitic transporto Pseudomonas aeruginosa is an important opportunist pathogen, frequently founded in patients with CF. In this patients, colonization starts with a non-mucoid form bacteria, afier this, bacteria turning to mucoid formo The objective of this work was to verify comparatively the production of some virulence factors by P. aeruginosa in mucoid and nonrnucoid strains isolated of CF patients and in strains isolated of others infections. The method to detect hemolysin used blood agar with 5% of sheep blood. The gelatin hydrolysis was used to detect gelatinase. Elastase production was observed used elastin 1 %. Pili production was analised by microhemagglutination method and alginate capsule by PCR method to detect genes algD e algU. Non-mucoid isolates of patients with FC and isolates others of clinical cases strains produces exoenzÍmes gelatinase and elastase in larger frequency than mucoid strains. Hemolysin production and presence of genes algD and algU was resembling on three bacterians groups. The gene algD was founded in ali studied strains and essay of microhemagglutination was not satisfactory to detect pili in P. aeruginosa strainsMestradoMicrobiologiaMestre em Ciências Biológica

Analise das caracteristicas da patogenicidade de linhagens de Escherichia coli causadoras de septicemia e sindrome de cabeça inchada em aves

Orientador: Wanderley Dias da SilveiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaDoutorad